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Acta Biochim Biophys Sin (Shanghai)
2020 Dec 11;5211:1215-1226. doi: 10.1093/abbs/gmaa114.
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X-box-binding protein 1 is required for pancreatic development in Xenopus laevis.
Yang J
,
Liu X
,
Yuan F
,
Liu J
,
Li D
,
Wei L
,
Wang X
,
Yuan L
.
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X-box-binding protein 1 (XBP1) is a protein containing the basic leucine zipper structure. It belongs to the cAMP-response element binding protein (CREB)/activating transcription factor transcription factor family. As the main transcription factor, spliced XBP1 (XBP1s) participates in many physiological and pathological processes and plays an important role in embryonic development. Previous studies showed that XBP1-knockout mice died because of pancreatic exocrine function deficiency, indicating that XBP1 plays an important role in pancreatic development. However, the exact role of XBP1 in pancreatic development remains unclear. This study aimed to investigate the role of XBP1 in the pancreatic development of Xenopus laevis embryos. Whole-mount in situ hybridization and quantitative real-time PCR results revealed that the expression levels of pancreatic progenitor marker genes pdx1, p48, ngn3, and sox9 were downregulated in XBP1s morpholino oligonucleotide (MO)-injected embryos. The expression levels of pancreatic exocrine and endocrine marker genes insulin and amylase were also downregulated. Through the overexpression of XBP1s, the phenotype and gene expressions were opposite to those in XBP1s MO-injected embryos. Luciferase and chromatin immunoprecipitation assays showed that XBP1s could bind to the XBP1-binding site in the foxa2 promoter. These results revealed that XBP1 is required in the pancreatic development of Xenopus laevis and might function by regulating foxa2.
Figure 1.
XBP1s expression in Xenopus laevis during embryonic development (A) Western blot analysis was used to detect XBP1s expression in different stages of X. laevis embryonic development (stage 4: cleavage stage; stage 11: gut stage; stage 18: neuroblast stage; stage 28 and stage 32: tailbud stage; stage 42: sputum stage). (B) Whole-mount in situ hybridization was used to detect the spatial expression of XBP1 mRNA. (C) Immunostaining was used to detect the spatial expression of XBP1 protein (md: mesoderm; ed: ectoderm; n: nerve; hd: head; cv: main vein; ph: anteriorkidney; mg: mucous gland; abd: abdomen; ht: heart; pa: pancreas).
Figure 2.
XBP1 knockdown inhibited embryonic development (A) Schematic diagram of the XBP1s MO mechanism of action. (B) XBP1s knockdown upon injection of 50 ng MO in the sputum stage (stage 42). Western blot analysis showed the knockdown efficiency. *P<0.05, n=3. (C) Effects of XBP1 knockdown on embryonic development in different stages.
Figure 3.
Effects of XBP1s knockdown on the germ layer formation (A) Whole-mount in situ hybridization was used to detect the changes in the germ layers at stage 11 after XBP1s knockdown. (BD) qPCR detection of the expressions of germ layer markers with XBP1s knockdown. *P<0.05, n=9.
Figure 4.
Effects of XBP1s knockdown on the development of pancreatic endocrine and exocrine cells (A) Immunohistochemical detection of the changes of the expression level of insulin after XBP1s knockdown. (B) qPCR was used to detect the expressions of pancreatic marker genes at stage 42 after XBP1s knockdown compared with the control group. *P<0.05, **P<0.01, n=9.
Figure 5.
Effects of XBP1s knockdown on the pancreatic precursor cell (A) In situ hybridization detection of the expression of pancreatic marker genes pdx1 and pft1a, liver marker gene hhex, and intestinal marker gene darmin at stage 37 after XBP1s knockdown. (B) qPCR detection of the changes in the expression levels of pancreatic, liver, intestinal, and other organ marker genes after XBP1s knockdown compared with the control group. *P<0.05, **P<0.01, n=9.
Figure 6.
Effect of XBP1 overexpression on Xenopus embryonic development Dexamethasone was added to the culture medium at stages 11, 15, 20, and 25 to activate XBP1s-GR expression and hence induce XBP1s overexpression in specific developmental stages.
Figure 7.
Effect of XBP1s overexpression on pancreatic development (A) Whole-mount in situ hybridization revealed higher expression level of insulin, and lower expression levels of hhex and darmin, when XBP1s was overexpressed at stage 11. (B) Immunohistochemical analysis revealed high expression of insulin after XBP1 overexpression.
Figure 8.
Effect of XBP1s knockdown on embryonic cell proliferation and apoptosis (A,B) TUNEL staining of the whole embryos in the control group (A) and XBP1 knockdown group (B) (blue, punctate, apoptotic cells). (C,D) pH3 staining of whole embryos in the control group (C) and XBP1 knockdown group (D) (red, punctate, proliferating cells). (E) Quantitative representation of the TUNEL and pH3 staining. *P<0.05.
Figure 9.
Transcriptional regulation of foxa2 by XBP1s (A) Bioinformatics analysis of the binding site of XBP1 in the foxa2 promoter region. (B) Luciferase assay was used to detect whether two binding sites of foxa2 are effective. These two possible binding regions were cloned into the pGL3-basic plasmid, and the 293T cells were co-transfected with the XBP1 plasmid and the binding site plasmid. (C) ChIP assay was used to detect the binding of XBP1 to the promoter region of foxa2 in vivo. XBP1s-myc mRNA was injected into the embryos, and the embryos were collected at the stage of neuroblasts. Then, the experiments were performed using myc antibodies, and primers were designed upstream and downstream for site 1 for PCR detection.
